Glycans of the early human yolk sac

Summary The pattern of glycan distribution in the early human yolk sac has been investigated using a panel of lectins. Two 6-week and one 8-week human yolk sacs, and one 8-week fetal liver from live, ectopic pregnancies were fixed and embedded in epoxy resin. Lectin histochemistry was carried out on sections of these tissues using 23 biotinylated lectins and an avidin-biotin peroxidase revealing system. Mesothelial surfaces expressed most subsets of N-glycans (other than high mannose types),N-acetyl-lactosamine, sialic acid, andα1,6-N-acetylgalactosamine. Endodermal surface and lateral membranes resembled those of mesothelium, but showed a preponderance ofα2,6-sialyl residues. Most intracellular granules contained N-glycan. There was a marked heterogeneity of granules in the endodermal cells, with different subsets varying in both staining and positional characteristics. The mesenchymal matrix bound most of the lectins used in the study, and expressed fucosyl residues which were also detected in the endothelium. Fetal liver parenchyma showed very similar staining patterns to those seen in the endoderm except for the distribution ofN-acetylglucosamine, which was sparse. Despite some common features, each germ cell layer had a distinct ‘glycotype’, with some saccharides showing extreme topographical restriction.     

Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on β-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that β-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with β-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous β-catenin. Steadystate levels and nuclear accumulation of β-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased β-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of β-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in β-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in β-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.     

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana . Four classes of cDNAs representing four distinct AspAT genes ( ASP1—ASP4 ) have been cloned from Arabidopsis . Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1—AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis . These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.     

Human C4 polymorphism: Pedigree analysis of qualitative,quantiative, and functional parameters as a basis for phenotype interpretations

Summary Ten families with 82 members were investigated for C4A- and B polymorphism in a blind trial. Phenotyping was done on neuraminidase treated sera by immunofixation and simulataneously by hemolytic overlay electrophoresis. In addition Rg, Ch, BF, C2, HLA-A, B, C, DR, and GLO were determined. After decoding the samples the reliability of blind typing was found to be 84.4% according to segregation patters. Inconsistencies occurred mostly when A 4, A 2, or A 92 were present. The detection of silent A*Q0 and B*Q0 alleles was more critical than that of difficult allotypes. The quantitation of the C4A/B ratio by densitometry of stained gels or by conventional immunochemical measurements of serum C4 level could not substantially improve the identification of A*Q0 or B*Q0. C4 dependent activity in radial diffusion hemolysis showed satisfactory correspondence with the number of expressed C4B alleles. At least three haplotypes with two C4A genes (duplicated A genes) were observed as ascertained from offspring analysis in accordance with the MHC segregation pattern. Individuals with the duplicated C4A gene (C4A*3. A*2. in the absence of any other expressed A allele or together with C4A*92) showed only partial inhibition of Rodgers antisera. Partial inhibition of Chido antisera was seen in individuals with C4B 2 (in the absence of other B allotypes). The findings support the hypothesis of at least two structural C4 loci. The also demonstrate the inconsistency of quantitative data in the recognition of silent alleles.     

Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

Sexual dimorphism and resource allocation in male and female shrubs of Simmondsia chinensis

Summary Desert populations of the evergreen dioecious shrub Simmondsia chinensis exhibit sex-related leaf and canopy dimorphisms not present in populations from more mesic coastal environments. Leaves on female shrubs have characteristically larger sizes, greater specific weights, and greater water-holding capacity than male leaves in desert habitats. In coastal scrub environments no significant difference is present, with leaf characteristics of both sexes similar to those of desert male shrubs. Desert female shrub canopies are typically relatively open with little mutual branch shading. In male shrubs canopies are more densely branched with considerable mutual shading of branches. Female plants allocate a greater proportion of their vegetative resources to leaves than do male plants. Considering total biomass, male plants allocate 10–15% of their resources (biomass, calories, glucose-equivalents, nitrogen, phosphorus) to reproductive tissues. Female allocation is dependent on seed set. At 100% seed set females would allocate 30–40% of their resources to reproduction, while female reproductive investment would equal that of males at approximately 30% seed set. Sexual dimorphism and the associated physiological characteristics in Simmondsia act as an alternative to differential habitat selection by male and female plants. Female plants respond to limited water resources in desert areas by increasing their efficiency in allocating limited resources to reproductive structures.     

The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315

Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H₍₃₎ resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H₍₃₎ proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93_L, in an `aromatic box' of essentially tryptophan-93_L, phenylalanine-34_H and tyrosine-34_L; asparagine-36_L and tyrosine-34_L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.     

Actin filaments in muscle: Pattern of myosin and tropomyosin/troponin attachments

X-ray patterns from lobster and crayfish muscles show very clear layer lines from the thin filaments, well separated from the myosin layer lines. The intensities in patterns from relaxed muscles include an important contribution from the regulatory proteins, and allow the arrangement of the troponin complexes to be deduced. Moreover, the troponin diffraction indirectly provides an accurate value for the pitch of the actin helix in relaxed muscle.In rigor, the attachment of cross-bridges modifies the intensities. These X-ray patterns support Reedy's (1968) concept that cross-bridges in rigor attach only to certain azimuths on the actin filaments (“target areas”); the 145 Å repeat of their origins on the thick filaments is not reflected in the pattern of attachment. Our calculations show that the observed intensities agree quantitatively with those expected for models based on such attachment, but depend significantly on the locations of the troponin complexes. The arrangement of the filament components is discussed in terms of design requirements. Our conclusions may be applicable to many other muscles, especially insect flight muscle and other invertebrate muscles.     

Predation by a water mite (Piona exigua) on enclosed populations of zooplankton

Short term, replicated experimental alteration of densities of a predatory water mite Piona exigua Viets, inside 0.45–0.475 m³ litre enclosures, revealed little evidence of the effects of predation on the number and relative abundance of the enclosed zooplankton species. Predation rates more closely approximated those estimated from single prey functional response experiments in the second experimental period (December) than in the first (March). In December Daphnia was the only susceptible taxon present in large numbers, whereas in March, Ceriodaphnia and Chydorus were also present. This result is consistent with laboratory findings that predation rates are lowered in the presence of more than one prey type.The difficulty of obtaining evidence for significant effects of these planktonic predators is in part due to changes in the preferred prey species in the diet of Piona depending on stage and sex of the mite and to aspects of experimental design. The wide variability between replicate enclosures at each predator density reduced the power of the statistical analyses used to test the null hypothesis. Enclosures with no predators are necessary to investigate the effects of enclosure on the zooplankton prey, since these effects may outweigh those due to predator consumption.     

Histochemical analysis of rat testicular glycoconjugates. 1. Subsets of N-linked saccharides in seminiferous tubules

Summary The distribution of N-linked glycans in rat testis has been probed using a panel of lectins derived fromGalanthus nivalis (snowdrop, GNA),Canavalia ensiformis (jack bean, Con A),Lens culinaris (lentil, LCA),Pisum sativum (garden pea, PSA) andPhaseolus vulgaris, erythro- and leucoagglutinins (kidney bean, ePHA and IPHA). Several classes of N-linked glycan were identified in the spermatogenic series, and during differentiation into spermatozoa they altered in both their pattern of distribution and relative abundance. A population of tetra-antennary, non-bisected, complex glycans, detected by IPHA, was lost during the transition from spermatogonia to spermatocytes, while high-mannose structures were acquired; these were most abundant in spermatocytes, as were bi- and tri-antennary complex, non-bisected glycans, the latter becoming increasingly abundant on acrosomes and spermatozoa. Their bisected counterparts were more generally expressed throughout spermatogenic cells, although marked localization onto acrosomes and nuclear caps was again seen. Transition from spermatocytes to spermatids involved mainly changes of the acrosomal granule and nuclear cap, which were carried through to the final stages of differentiation. Sertoli cell surfaces and cytoplasmic granules showed a high level of N-glycan expression.